The need for new antimicrobials is increasingly urgent. The rate of multidrug resistant pathogens continues to increase, leading to significant morbidity and mortality throughout the world. Furthermore, the current pipeline for new antimicrobials remains very narrow. The Infectious Diseases Society of America has identified in their Bad Bugs, No Drugs campaign, a group of pathogens that have become increasingly resistant to current antibiotics. This group includes the Gram-negative pathogens Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. A new paradigm in antibiotic discovery and design has recently been shown effective against numerous bacteria. This new approach is based on a platform technology called peptide-phosphorodiamidate mopholino oligomers (PPMOs). PPMOs are synthetic DNA mimics that bind to RNA in a sequence-specific, antisense manner and inhibit expression of target bacterial genes. PPMOs have already been used successfully to kill a variety of bacterial pathogens including the Gram-negative bacteria Escherichia coli, Salmonella typhimurium, Burkholderia cepacia complex and Acinetobacter baumannii. PPMOS are bactericidal in culture, and reduce bacteremia and improve survival in animal models of infection. PPMOs are more potent than many traditional antibiotics such as ampicillin. The goal of this project is to develop PPMOs for therapeutic use against the multidrug resistant pathogens Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae. The specific aims are to design, produce and screen PPMOS against various gene targets in these multidrug-resistant pathogens. The experimental approach will be to target virulence properties in Acinetobacter and Pseudomonas, and essential or antibiotic resistance genes in Klebsiella. Lead compounds that are effective during our screening phase will then be tested for efficacy in animal models of infection. This technology provides a methodological advantage because many PPMOs can be rapidly synthesized and simultaneously tested against numerous targets. This allows for the possibility of targeting multiple genes in a single organism or the development of cocktails of PPMOs that target multiple pathogens. This project will identify lead target PPMOs in these medically important Gram- negative pathogens that can be moved forward to pre-clinical and clinical studies.